Compositions comprising bacterial strains

ABSTRACT

The invention provides compositions comprising one or more bacterial strains for use in a method of reducing the level of Enterobacteriaceae in the gastrointestinal tract.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.16/022,577, filed Jun. 28, 2018, which is a continuation of U.S.application Ser. No. 15/916,205, filed Mar. 8, 2018 which is acontinuation of International Application No. PCT/GB2017/052076, filedJul. 13, 2017, which claims priority to Great Britain Application No.1612190.7, filed Jul. 13, 2016; Great Britain Application No. 1616018.6,filed Sep. 20, 2016; Great Britain Application No. 1616016.0, filed Sep.20, 2016; Great Britain Application No. 1703548.6, filed Mar. 6, 2017;and Great Britain Application No. 1703552.8, filed Mar. 6, 2017; all ofwhich are hereby incorporated by reference in their entirety. Further,all publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 19, 2020, isnamed 49455_723_303_Sequence_Listing.txt and is 12,288 bytes in size.

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterialstrains isolated from the mammalian digestive tract and the use of suchcompositions in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be sterile in utero, but it is exposedto a large variety of maternal and environmental microbes immediatelyafter birth. Thereafter, a dynamic period of microbial colonization andsuccession occurs, which is influenced by factors such as delivery mode,environment, diet and host genotype, all of which impact upon thecomposition of the gut microbiota, particularly during early life.Subsequently, the microbiota stabilizes and becomes adult-like [1]. Thehuman gut microbiota contains more than 1500 different phylotypesdominated in abundance levels by two major bacterial divisions (phyla),the Bacteroidetes and the Firmicutes [2-3]. The successful symbioticrelationships arising from bacterial colonization of the human gut haveyielded a wide variety of metabolic, structural, protective and otherbeneficial functions. The enhanced metabolic activities of the colonizedgut ensure that otherwise indigestible dietary components are degradedwith release of by-products providing an important nutrient source forthe host and additional health benefits. Similarly, the immunologicalimportance of the gut microbiota is well-recognized and is exemplifiedin germfree animals which have an impaired immune system that isfunctionally reconstituted following the introduction of commensalbacteria [4-6].

Dramatic changes in microbiota composition have been documented ingastrointestinal disorders such as inflammatory bowel disease (IBD). Forexample, the levels of Clostridium cluster XIVa and Clostridium clusterXI (F. prausnitzii) bacteria are reduced in IBD patients whilst numbersof E. coli are increased, suggesting a shift in the balance of symbiontsand pathobionts within the gut [7-11].

In recognition of the potential positive effect that certain bacterialstrains may have on the animal gut, various strains have been proposedfor use in the treatment of various diseases (see, for example,[12-15]). A number of strains, including mostly Lactobacillus andBifidobacterium strains, have been proposed for use in treating variousbowel disorders (see [16] for a review). Strains of the genus Blautiahave also been proposed for use in modulating the microbial balance ofthe digestive ecosystem in IBS patients (WO 01/85187). However, therelationship between different bacterial strains and different diseases,and the precise effects of particular bacterial strains on the gut andat a systemic level and on any particular types of diseases, are poorlycharacterised.

There is a requirement for the potential effects of gut bacteria to becharacterised so that new therapies using gut bacteria can be developed.

US 2010/0247489 describes the use of mineral nutrients to treatdigestive disorders. US′789 proposes optionally also using numerousdifferent genera of bacteria, including Enterobacteriaceae, to preventand reduce gas formation in the colon, so this document suggests thatincreasing Enterobacteriaceae spp. in the gastrointestinal tract may beemployed to treat certain diseases. US′789 does not discuss any methodsfor reducing Enterobacteriaceae spp.

WO 2016/086206 suggests that bacteria in the order Clostridiales,including Enterbacteriaceace spp., can be used to treat or preventdysbiosis. There is no suggestion in WO′206 that reducing the level ofEnterobacteriaceace spp. in the gastrointestinal tract can be used totreat diseases, nor any suggestion regarding how a reduction in thelevel of Enterbacteriaceace spp. might be achieved.

WO 2012/142605 proposes that it may be possible to treat a number ofdifferent diseases with a combination of microorganisms. WO′605 suggestsa large number of possible bacterial species that could be employed, butthere is no teaching in WO′605 as to how any of the proposed bacterialspecies could be used to treat any of the diseases proposed.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating and preventingdiseases associated with Enterobacteriaceae, in particular E. coli. Inparticular, the inventors have identified that bacterial strains fromthe genus Blautia can be effective for reducing the level ofEnterobacteriaceae in the gastrointestinal tract. As described in theexamples, oral administration of compositions comprising Blautiahydrogenotrophica may reduce the level of Enterobacteriaceae in thegastrointestinal tract, including E. coli, and may treat or preventdiseases associated with Enterobacteriaceae or E. coli. Therefore, in afirst embodiment, the invention provides a composition comprising abacterial strain of the genus Blautia, for use in a method of reducingthe level of Enterobacteriaceae in the gastrointestinal tract.

In preferred embodiments of all aspects of the invention, theEnterobacteriaceae is E. coli. In preferred embodiments of all aspectsof the invention, the bacterial strain is of Blautia hydrogenotrophicaand is preferably the bacterium deposited under accession number DSM10507/14294.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in a methodof treating or preventing a disease associated with Enterobacteriaceaeinfection, such as E. coli infection. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhoea, gastroenteritis, urinary tract infection or neonatalmeningitis. In certain embodiments, the compositions of the inventionare for use in treating or preventing Enterobacteriaceae infection, suchas E. coli infection.

In further preferred embodiments, the compositions of the invention arefor use in reducing the level of Enterobacteriaceae in thegastrointestinal tract, preferably the level of E. coli, in thetreatment or prevention of IBS, Crohn's disease, ulcerative colitis,functional dyspepsia, diarrhoea, gastroenteritis, urinary tractinfection or neonatal meningitis.

The inventors have developed new therapies for treating and preventingdiarrhoea. In particular, the inventors have identified that bacterialstrains from the genus Blautia can be effective for reducing diarrhoea.As described in the examples, oral administration of compositionscomprising Blautia hydrogenotrophica may reduce diarrhoea in patientshaving irritable bowel syndrome (IBS).

In preferred embodiments of the invention, the bacterial strain in thecomposition is of Blautia hydrogenotrophica. Closely related strains mayalso be used, such as bacterial strains that have a 16s rRNA sequencethat is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical tothe 16s rRNA sequence of a bacterial strain of Blautiahydrogenotrophica. Preferably, the bacterial strain has a 16s rRNAsequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%identical to SEQ ID NO:5. Most preferably, the bacterial strain in thecomposition is the Blautia hydrogenotrophica strain deposited underaccession number DSM 10507/14294.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia stercoris. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia stercoris. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 3. Preferably, thesequence identity is to SEQ ID NO:3. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:3.

In further embodiments of the invention, the bacterial strain in thecomposition is of Blautia wexlerae. Closely related strains may also beused, such as bacterial strains that have a 16s rRNA sequence that is atleast 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNAsequence of a bacterial strain of Blautia wexlerae. Preferably, thebacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or 4. Preferably, thesequence identity is to SEQ ID NO:4. Preferably, the bacterial strainfor use in the invention has the 16s rRNA sequence represented by SEQ IDNO:4.

In certain embodiments, the composition of the invention is for oraladministration. Oral administration of the strains of the invention canbe effective for reducing the level of Enterobacteriaceae in thegastrointestinal tract. Also, oral administration is convenient forpatients and practitioners and allows delivery to and/or partial ortotal colonisation of the intestine.

In certain embodiments, the composition of the invention comprises oneor more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises abacterial strain that has been lyophilised. Lyophilisation is aneffective and convenient technique for preparing stable compositionsthat allow delivery of bacteria, and is shown to provide effectivecompositions in the examples.

In certain embodiments, the invention provides a food product comprisingthe composition as described above.

In certain embodiments, the invention provides a vaccine compositioncomprising the composition as described above.

Additionally, the invention provides a method of reducing the level ofEnterobacteriaceae in the gastrointestinal tract and thereby treating orpreventing diseases associated with Enterobacteriaceae, comprisingadministering a composition comprising a bacterial strain of the genusBlautia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Measurement of BH population by qPCR, showing an increase in BHat days 14 and 28 for IBS HMI rats receiving the BH lyophilisate.

FIG. 2: Dosing study in HIM rats—RT-PCR quantification ofB.hydrogenotrophica in fecal samples of Healthy HIM rats receivingdifferent concentration of the bacterial species.

FIG. 3: Transit time of B. hydrogenotrophica after oral administration(10⁹/day) to healthy HIM rats.

FIG. 4: Comparison of B. hydrogenotrophica levels found in fecal andcaecal samples of healthy HIM rats (RT-PCR quantification) after 14 daysadministration—B. hydrogenotrophica administrated at 10¹⁰/day/rat.

FIG. 5: Effect of B. hydrogenotrophica administration on faecalmicrobiota composition of healthy-human microbiota-associated rats.Administration of 10¹⁰ B. hydrogenotrophica/day for 14 days (resultsfrom 2 healthy human faecal microbiota (20 HIM rats)—qPCR analysis).

FIG. 6: Impact of B. hydrogenotrophica administration on the microbialpopulations in IBS-HIM rats.

FIG. 7A: Change in levels of patient microbiota Blautiahydrogenotrophica during and following Blautix treatment.

FIG. 7B: Change in levels of patient microbiota Enterobacteria duringand following Blautix treatment.

FIGS. 8A-8C: Hydrogen breath test Cmax results for day 1, day 2, day 15and day 16 for IBS patients treated with Blautix (FIG. 8A) and IBSpatients treated with placebo (FIG. 8B). FIG. 8C is a graph comparingthe percentage of Blautix treated patients with a reduction in hydrogenbetween the mean of days 15 and 16 and the mean of days 1 and 2 to thepercentage of placebo treated patients with a reduction in hydrogenbetween these time points.

FIG. 9A: Hydrogen uncorrected and hydrogen corrected breath test paireddata for day 1 and day 15 for Blautix treated IBS patients; FIG. 9B:graph comparing mean hydrogen uncorrected breath test results for day 1and day 15 for the Blautix treatment group; FIG. 9C: graph comparingmean hydrogen corrected breath test results for day 1 and day 15 for theBlautix treatment group.

FIG. 10A: Hydrogen uncorrected and hydrogen corrected breath test paireddata for day 1 and day 15 for placebo treated IBS patients; FIG. 10B:graph comparing mean hydrogen uncorrected breath test results for day 1and day 15 for the placebo group; FIG. 10C: graph comparing meanhydrogen corrected breath test results for day 1 and day 15 for theplacebo group.

FIGS. 11A-11B: Graphs comparing the mean hydrogen breath test resultsfrom day 1 and day 15 for the Bautix treatment group (Verum) and placebogroup (FIG. 11A: uncorrected hydrogen;

FIG. 11B: corrected hydrogen).

FIG. 12: qPCR evaluation of B. hydrogenotrophica population in faecalsamples of IBS-HMA rats treated or not with a composition comprising B.hydrogenotrophica (BlautiX) for 28 days.

FIG. 13: Bacteria enumeration in IBS HMA-rat faecal samples after B.hydrogenotrophica (BlautiX) administration versus control solution.

FIG. 14: Changes in patient symptoms during dosing period (days 1-16) ofPhase I clinical trial.

FIG. 15: Changes in patient symptoms during washout period of Phase Iclinical trial.

DISCLOSURE OF THE INVENTION Bacterial Strains

The compositions of the invention comprise a bacterial strain of thegenus Blautia. The examples demonstrate that bacteria of this genus areuseful for reducing the level of Enterobacteriaceae in thegastrointestinal tract, in particular E. coli. The preferred bacterialstrains are of the species Blautia hydrogenotrophica, Blautia stercorisand Blautia wexlerae. Other preferred bacterial strains for use in theinvention are Blautia producta, Blautia coccoides and Blautia hansenii.Most preferred is Blautia hydrogenotrophica, particularly the bacteriumdeposited under accession number DSM 10507/14294.

Examples of Blautia strains for use in the invention include Blautiahydrogenotrophica, B. stercoris, B. faecis, B. coccoides, B. glucerasea,B. hansenii, B. luti, B. producta, B. schinkii and B. wexlerae. TheBlautia species are Gram-reaction-positive, non-motile bacteria that maybe either coccoid or oval and all are obligate anaerobes that produceacetic acid as the major end product of glucose fermentation [17].Blautia may be isolated from the human gut, althoughB. producta wasisolated from a septicaemia sample.

Blautia hydrogenotrophica (previously known as Ruminococcushydrogenotrophicus) has been isolated from the guts of mammals, isstrictly anaerobic, and metabolises H₂/CO₂ to acetate, which may beimportant for human nutrition and health. The type strain of Blautiahydrogenotrophica is S5a33=DSM 10507=JCM 14656. The GenBank accessionnumber for the 16S rRNA gene sequence of Blautia hydrogenotrophicastrain S5a36 is X95624.1 (disclosed herein as SEQ ID NO:5). Thisexemplary Blautia hydrogenotrophica strain is described in [17] and[18]. The S5a33 strain and the S5a36 strain correspond to two subclonesof a strain isolated from a faecal sample of a healthy subject. Theyshow identical morphology, physiology and metabolism and have identical16S rRNA sequences. Thus, in some embodiments, the Blautiahydrogenotrophica for use in the invention has the 16S rRNA sequence ofSEQ ID NO:5.

The Blautia hydrogenotrophica bacterium deposited under accession numberDSM 10507 and also under accession number DSM 14294 was tested in theexamples and is also referred to herein as strain BH. Strain BH wasdeposited with the Deutsche Sammlung von Mikroorganismen [GermanMicroorganism Collection] (Mascheroder Weg 1b, 38124 Braunschweig,Germany) in 26 Jan. 1996 as “Ruminococcus hydrogenotrophicus” underaccession number DSM 10507 and also under accession number DSM 14294 as“S5a33” on 14 May 2001. The depositor was INRA Laboratoire deMicrobiologie CR de Clermont-Ferrand/Theix 63122 Saint GenesChampanelle, France. Ownership of the deposits has passed to 4D PharmaPlc by way of assignment. The DSM 14294 and DSM 10507 deposits were madeunder the terms of the Budapest Treaty. Maintenance of a viable cultureis assured for 30 years from the date of deposit. All restrictions onthe availability to the public of microorganisms deposited as DSM 14294and DSM 10507 will be irrevocably removed upon the granting of a patentfor this application.

The GenBank accession number for the 16S rRNA gene sequence of Blautiastercoris strain GAM6-1^(T) is HM626177 (disclosed herein as SEQ IDNO:1). An exemplary Blautia stercoris strain is described in [19]. Thetype strain of Blautia wexlerae is WAL 14507=ATCC BAA-1564=DSM 19850[17]. The GenBank accession number for the 16S rRNA gene sequence ofBlautia wexlerae strain WAL 14507 T is EF036467 (disclosed herein as SEQID NO:2). This exemplary Blautia wexlerae strain is described in [17].

A preferred Blautia stercoris strain is the strain deposited underaccession number NCIMB 42381, which is also referred to herein as strain830. A 16S rRNA sequence for the 830 strain is provided in SEQ ID NO:3.Strain 830 was deposited with the international depositary authorityNCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GTBiologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,Scotland) on 12 Mar. 2015 as “Blautia stercoris 830” and was assignedaccession number NCIMB 42381. GT Biologics Ltd. subsequently changed itsname to 4D Pharma Research Limited. The NCIMB 42381 deposit was madeunder the terms of the Budapest Treaty. Maintenance of a viable cultureis assured for 30 years from the date of deposit. All restrictions onthe availability to the public of the deposited microorganism will beirrevocably removed upon the granting of a patent for this application.

A preferred Blautia wexlerae strain is the strain deposited underaccession number NCIMB 42486, which is also referred to herein as strainMRX008. A 16S rRNA sequence for the MRX008 strain is provided in SEQ IDNO:4. Strain MRX008 was deposited with the international depositaryauthority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland)by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen,AB25 2ZS, Scotland) on 16 Nov. 2015 as “Blaqutia/Ruminococcus MRx0008”and was assigned accession number NCIMB 42486. The NCIMB 42486 depositwas made under the terms of the Budapest Treaty. Maintenance of a viableculture is assured for 30 years from the date of deposit. Allrestrictions on the availability to the public of the depositedmicroorganism will be irrevocably removed upon the granting of a patentfor this application.

Bacterial strains closely related to the strain tested in the examplesare also expected to be effective for reducing the level ofEnterobacteriaceae in the gastrointestinal tract, in particular E. coli.In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of a bacterial strain ofBlautia hydrogenotrophica. Preferably, the bacterial strain for use inthe invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5.

In certain embodiments, the bacterial strain for use in the inventionhas a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%or 99.9% identical to the 16s rRNA sequence of a bacterial strain ofBlautia stercoris. Preferably, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to SEQ ID NO:1 or SEQ ID NO:3. Preferably,the sequence identity is to SEQ ID NO:3. Preferably, the bacterialstrain for use in the invention has the 16s rRNA sequence represented bySEQ ID NO:3. In certain embodiments, the bacterial strain for use in theinvention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%,99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterialstrain of Blautia wexlerae. Preferably, the bacterial strain for use inthe invention has a 16s rRNA sequence that is at least 95%, 96%, 97%,98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:4.Preferably, the sequence identity is to SEQ ID NO:4. Preferably, thebacterial strain for use in the invention has the 16s rRNA sequencerepresented by SEQ ID NO:4.

Bacterial strains that are biotypes of the bacterium deposited underaccession number DSM 10507/14294 or biotypes of the bacteria depositedunder accession numbers NCIMB 42381 and NCIMB 42486 are also expected tobe effective for reducing the level of Enterobacteriaceae in thegastrointestinal tract, in particular E. coli. A biotype is a closelyrelated strain that has the same or very similar physiological andbiochemical characteristics.

Strains that are biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and that are suitablefor use in the invention may be identified by sequencing othernucleotide sequences for a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486. For example, substantiallythe whole genome may be sequenced and a biotype strain for use in theinvention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%sequence identity across at least 80% of its whole genome (e.g. acrossat least 85%, 90%, 95% or 99%, or across its whole genome). For example,in some embodiments, a biotype strain has at least 98% sequence identityacross at least 98% of its genome or at least 99% sequence identityacross 99% of its genome. Other suitable sequences for use inidentifying biotype strains may include hsp60 or repetitive sequencessuch as BOX, ERIC, (GTG)₅, or REP or [20]. Biotype strains may havesequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenceidentity to the corresponding sequence of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andcomprises a 16S rRNA sequence that is at least 99% identical (e.g. atleast 99.5% or at least 99.9% identical) to SEQ ID NO:5. In someembodiments, a biotype strain has a sequence with at least 97%, 98%,99%, 99.5% or 99.9% sequence identity to the corresponding sequence ofthe Blautia hydrogenotrophica strain deposited as DSM 10507/14294 andhas the 16S rRNA sequence of SEQ ID NO:5.

Alternatively, strains that are biotypes of a bacterium deposited underaccession number DSM 10507/14294, NCIMB 42381 or NCIMB 42486 and thatare suitable for use in the invention may be identified by using theaccession number DSM 10507/14294 deposit, the accession number NCIMB42381 deposit, or the accession number NCIMB 42486 deposit, andrestriction fragment analysis and/or PCR analysis, for example by usingfluorescent amplified fragment length polymorphism (FAFLP) andrepetitive DNA element (rep)-PCR fingerprinting, or protein profiling,or partial 16S or 23s rDNA sequencing. In preferred embodiments, suchtechniques may be used to identify other Blautia hydrogenotrophica,Blautia stercoris or Blautia wexlerae strains.

In certain embodiments, strains that are biotypes of a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 and that are suitable for use in the invention are strains thatprovide the same pattern as a bacterium deposited under accession numberDSM 10507/14294, NCIMB 42381 or NCIMB 42486 when analysed by amplifiedribosomal DNA restriction analysis (ARDRA), for example when usingSau3AI restriction enzyme (for exemplary methods and guidance see, forexample,[21]). Alternatively, biotype strains are identified as strainsthat have the same carbohydrate fermentation patterns as a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486.

Other Blautia strains that are useful in the compositions and methods ofthe invention, such as biotypes of a bacterium deposited under accessionnumber DSM 10507/14294, NCIMB 42381 or NCIMB 42486, may be identifiedusing any appropriate method or strategy, including the assays describedin the examples. For instance, strains for use in the invention may beidentified by culturing bacteria and administering to rats to test inthe distension assay. In particular, bacterial strains that have similargrowth patterns, metabolic type and/or surface antigens to a bacteriumdeposited under accession number DSM 10507/14294, NCIMB 42381 or NCIMB42486 may be useful in the invention. A useful strain will havecomparable microbiota modulatory activity to the DSM 10507/14294, NCIMB42381 or NCIMB 42486 strain. In particular, a biotype strain will elicitcomparable effects on Enterobacteriaceae to the effects shown in theExamples, which may be identified by using the culturing andadministration protocols described in the Examples.

A particularly preferred strain of the invention is the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294. This is the exemplary BH strain tested in the examples andshown to be effective for reducing the level of Enterobacteriaceae inthe gastrointestinal tract, in particular E. coli. Therefore, theinvention provides a cell, such as an isolated cell, of the Blautiahydrogenotrophica strain deposited under accession number DSM10507/14294, or a derivative thereof, for use in therapy, in particularfor the diseases described herein.

A derivative of the strain deposited under accession number DSM10507/14294, NCIMB 42381 or NCIMB 42486 may be a daughter strain(progeny) or a strain cultured (subcloned) from the original. Aderivative of a strain of the invention may be modified, for example atthe genetic level, without ablating the biological activity. Inparticular, a derivative strain of the invention is therapeuticallyactive. A derivative strain will have comparable microbiota modulatoryactivity to the original DSM 10507/14294, NCIMB 42381 or NCIMB 42486strain. In particular, a derivative strain will elicit comparableeffects on Enterobacteriaceae to the effects shown in the Examples,which may be identified by using the culturing and administrationprotocols described in the Examples. A derivative of the DSM 10507/14294strain will generally be a biotype of the DSM 10507/14294 strain. Aderivative of the NCIMB 42381 strain will generally be a biotype of theNCIMB 42381 strain. A derivative of the NCIMB 42486 strain willgenerally be a biotype of the NCIMB 42486 strain.

References to cells of the Blautia hydrogenotrophica strain depositedunder accession number DSM 10507/14294 encompass any cells that have thesame safety and therapeutic efficacy characteristics as the strainsdeposited under accession number DSM 10507/14294, and such cells areencompassed by the invention. References to cells of the Blautiastercoris strain deposited under accession number NCIMB 42381 encompassany cells that have the same safety and therapeutic efficacycharacteristics as the strains deposited under accession number NCIMB42381, and such cells are encompassed by the invention. References tocells of the Blautia wexlerae strain deposited under accession numberNCIMB 42486 encompass any cells that have the same safety andtherapeutic efficacy characteristics as the strains deposited underaccession number NCIMB 42486, and such cells are encompassed by theinvention.

In preferred embodiments, the bacterial strains in the compositions ofthe invention are viable and capable of partially or totally colonisingthe intestine.

Therapeutic Uses

In certain embodiments, the compositions of the invention are for use inreducing the level of Enterobacteriaceae in the gastrointestinal tract.Increased levels of Enterobacteriaceae in the gastrointestinal tract areassociated with numerous pathological conditions and diseases, and theexamples demonstrate that the compositions of the invention may beeffective for reducing the level of Enterobacteriaceae in thegastrointestinal tract.

In preferred embodiments of all aspects of the invention, theEnterobacteriaceae is E. coli. Therefore, preferably the compositions ofthe invention are for use in reducing the level of E. coli in thegastrointestinal tract.

In certain embodiments, the Enterobacteriaceae is a pathogenic strain,such as E. coli O157:H7, O104:H4, O121, O26, O103, O111, O145, O104:H21,or O104:H4.

In alternative embodiments, the Enterobacteriaceae is a commensal ornon-pathogenic strain. Increased levels of such Enterobacteriaceae maycontribute to conditions such as IBS and Crohn's disease, and theexamples demonstrate that the compositions of the invention may have areducing effect on Enterobacteriaceae in the context of IBS.

In certain embodiments, the compositions of the invention are for use intreating or preventing a disease associated with Enterobacteriaceaeinfection, such as E. coli infection. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhoea, gastroenteritis, urinary tract infection or neonatalmeningitis. In such embodiments the Enterobacteriaceae may be apathogenic strain.

In certain embodiments, the compositions of the invention are for use intreating or preventing a disease associated with increased levels ofEnterobacteriaceae, such as E. coli. In certain embodiments, thecompositions of the invention are for use in treating or preventingdiarrhoea, gastroenteritis, urinary tract infection or neonatalmeningitis. In such embodiments, the Enterobacteriaceae may be acommensal or non-pathogenic strain.

In some embodiments, the pathogenesis of the disease or conditionaffects the intestine. In some embodiments, the pathogenesis of thedisease or condition does not affect the intestine. In some embodiments,the pathogenesis of the disease or condition is not localised at theintestine. In some embodiments, the treating or preventing occurs at asite other than at the intestine. In some embodiments, the treating orpreventing occurs at the intestine and also at a site other than at theintestine. In certain embodiments, the disease or condition is systemic.

In certain embodiments, the compositions of the invention are for use intreating or preventing Enterobacteriaceae infection, such as E. coliinfection. In preferred embodiments, the compositions of the inventionare for use in treating or preventing infection of the gastrointestinaltract, and in particular the caecum. In such embodiments theEnterobacteriaceae may be a pathogenic strain.

In some embodiments, the level of Enterobacteriaceae is reduced in stoolin the subject. In some embodiments, the level of Enterobacteriaceae isreduced in a stool sample from the subject. In some embodiments, thelevel of Enterobacteriaceae is reduced in the distal gut of the subject.In some embodiments, the level of Enterobacteriaceae is reduced in thecaecum. In some embodiments, the level of Enterobacteriaceae is reducedin the colon. In some embodiments, the level of Enterobacteriaceae isreduced in the rectum. In some embodiments, the level ofEnterobacteriaceae is reduced in the small intestine.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in thetreatment of infections caused by pathogenic Enterobacteriaceae.

In preferred embodiments, the invention provides a compositioncomprising a bacterial strain of the genus Blautia, for use in thetreatment of E. coli infection.

In preferred embodiments, the compositions of the invention are for usein reducing the level of Enterobacteriaceae in the gastrointestinaltract, preferably the level of E. coli, in the treatment or preventionof a disease associated with increased levels of Enterobacteriaceae,such as IBS, Crohn's disease, ulcerative colitis, functional dyspepsia,diarrhoea, gastroenteritis, urinary tract infection or neonatalmeningitis. Enterobacteriaceae and in particular E. coli are known to bepotential triggers for, or known to exacerbate, Crohn's disease andulcerative colitis [22-24], so the effect shown in the examples for thecompositions of the invention may be beneficial in the treatment ofthese conditions.

In certain embodiments, the compositions of the invention are for use intreating or preventing IBS, Crohn's disease, ulcerative colitis,functional dyspepsia, diarrhoea, gastroenteritis, urinary tractinfection or neonatal meningitis by reducing the level ofEnterobacteriaceae in the gastrointestinal tract.

In preferred embodiments, the compositions of the invention comprise thebacterium deposited under accession number DSM 10507/14294 and are foruse in reducing the level of Enterobacteriaceae in the gastrointestinaltract in the treatment of Crohn's disease, ulcerative colitis,functional dyspepsia, or most preferably IBS. In further preferredembodiments, the compositions of the invention comprise the bacteriumdeposited under accession number DSM 10507/14294 and are for use intreating or preventing Crohn's disease, ulcerative colitis, functionaldyspepsia, or most preferably IBS by reducing the level ofEnterobacteriaceae in the gastrointestinal tract.

In certain embodiments, the composition is administered in combinationwith an antibiotic, such as a fluoroquinolone, azithromycin,ciprofloxacin or rifaximin. In certain embodiments, the composition isadministered in combination with a dehydration treatment such as oralrehydration solution. The composition of the invention may beadministered at the same time as the antibiotic or dehydrationtreatment, or sequentially with the antibiotic or dehydration treatment.

In certain embodiments, the compositions are for use in patients thatexhibit, or are expected to exhibit, increased levels ofEnterobacteriaceae in the gastrointestinal tract, for example, whencompared to a healthy subject, or a population of healthy subjects.

In certain embodiments, the compositions of the invention are for use ina method of treating, preventing or reducing colonisation of thegastrointestinal tract by Enterobacteriaceae.

In some embodiments, the composition of the invention is for use in amethod of reducing the level of Enterobacteriaceae in thegastrointestinal tract in a subject having an increased level ofhydrogen in their breath relative to a healthy subject. In someembodiments, the composition of the invention is for use in reducing thehydrogen level in the breath of a subject exhibiting or who is expectedto exhibit an increased level of Enterobacteriaceae in thegastrointestinal tract. The subject is preferably a subject diagnosed ashaving IBS, Crohn's disease, ulcerative colitis, functional dyspepsia,diarrhoea, gastroenteritis, urinary tract infection or neonatalmeningitis and/or an infection caused by pathogenic Enterobacteriaceae,for example, E. coli. The examples show that treatment with acomposition of the invention reduces the level of hydrogen detected inhydrogen breath tests. Accordingly, the hydrogen levels are preferablyassessed using a hydrogen breath test. The hydrogen breath test is wellknown in the art and so the skilled person will know how to conduct sucha test. In some embodiments, the patient is administered lactulose asthe substrate for the test.

The hydrogen breath test is also a useful tool for monitoring theeffectiveness or likely effectiveness of reducing the level ofEnterobacteriaceae and of treatment or prevention using a composition ofthe invention. For example, a reduction in the level of hydrogendetected in a subject's breath following treatment with a composition ofthe invention may indicate that the treatment is having a reducing,therapeutic or preventative effect. Accordingly, in some embodiments themethods and uses of the invention further comprise monitoring thehydrogen level in a subject's breath during and/or following treatmentwith a composition of the invention and thereby assessing theeffectiveness or likely effectiveness of reducing, treatment orprevention. For example, hydrogen levels may be monitored at one or more(e.g. 1, 2, 3, 4 or more than 4) times, for example, including beforetreatment, at the start of treatment, during treatment, at the end oftreatment and/or following treatment, as desired. In some embodiments,the level of hydrogen in the subject's breath at the end and/orfollowing the dosing period (during which the composition isadministered to the subject) is compared to the level at the startand/or before the dosing period and a reduction in the level indicatesthe effectiveness or likely effectiveness of the reducing, treatment orprevention. For example, in embodiments in which the dosing period is 16days, it may be desirable to take measurements at day 1 and day 16, orfor example at day 1, day 2, day 15 and day 16. In some embodiments,multiple measurements are taken and the mean of those measurementsobtained (for example, the mean of day 1 and day 2 and the mean of day15 and day 16). In some embodiments, a reduction in at least 40 ppm inthe hydrogen level Cmax indicates that the reducing, treatment orprevention is effective or likely to be effective. In some embodiments,the hydrogen level in the subject's breath is measured only once, forexample, at the end of or following treatment, and the finding that thelevel is at or close to a predetermined level is indicative that thereducing, treatment or prevention is likely to have been effective. Thehydrogen breath test is a standard assay and so predetermined levels areknown in the art.

Treatment or prevention may refer to, for example, an alleviation of theseverity of symptoms or a reduction in the frequency of exacerbations orthe range of triggers that are a problem for the patient.

Levels of Enterobacteriaceae May be Detected in Faeces from a Patient,Using Standard Techniques,

such as the qPCR techniques used in the examples.

Modes of administration Preferably, the compositions of the inventionare to be administered to the gastrointestinal tract in order to enabledelivery to and/or partial or total colonisation of the intestine withthe bacterial strain of the invention. Generally, the compositions ofthe invention are administered orally, but they may be administeredrectally, intranasally, or via buccal or sublingual routes.

In certain embodiments, the compositions of the invention may beadministered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may beadministered as a suppository, such as a rectal suppository, for examplein the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g.suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soapglycerin composition.

In certain embodiments, the composition of the invention is administeredto the gastrointestinal tract via a tube, such as a nasogastric tube,orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneousendoscopic gastrostomy (PEG), or a port, such as a chest wall port thatprovides access to the stomach, jejunum and other suitable access ports.

The compositions of the invention may be administered once, or they maybe administered sequentially as part of a treatment regimen. In certainembodiments, the compositions of the invention are to be administereddaily. The examples demonstrate that daily administration providessuccessfully colonisation and clinical benefits.

In certain embodiments, the compositions of the invention areadministered regularly, such as daily, every two days, or weekly, for anextended period of time, such as for at least one week, two weeks, onemonth, two months, six months, or one year. The examples demonstratethat BH administration may not result in permanent colonisation of theintestines, so regular administration for extended periods of time mayprovide greater therapeutic benefits.

In some embodiments the compositions of the invention are administeredfor 7 days, 14 days, 16 days, 21 days or 28 days or no more than 7 days,14 days, 16 days, 21 days or 28 days. For example, in some embodimentsthe compositions of the invention are administered for 16 days.

In certain embodiments of the invention, treatment according to theinvention is accompanied by assessment of the patient's gut microbiota.Treatment may be repeated if delivery of and/or partial or totalcolonisation with the strain of the invention is not achieved such thatefficacy is not observed, or treatment may be ceased if delivery and/orpartial or total colonisation is successful and efficacy is observed.

In certain embodiments, the composition of the invention may beadministered to a pregnant animal, for example a mammal such as a humanin order to prevent elevated levels of Enterobacteriaceae developing inher child in utero and/or after it is born.

The compositions of the invention may be administered to a patient thathas been diagnosed with elevated levels of Enterobacteriaceae or adisease or condition associated with elevated levels ofEnterobacteriaceae, or that has been identified as being at risk ofelevated levels of Enterobacteriaceae. The compositions may also beadministered as a prophylactic measure to prevent the development ofelevated levels of Enterobacteriaceae in a healthy patient.

The compositions of the invention may be administered to a patient thathas been identified as having an abnormal gut microbiota. For example,the patient may have reduced or absent colonisation by Blautia, and inparticular Blautia hydrogenotrophica, Blautia stercoris or Blautia wexlerae.

The compositions of the invention may be administered as a food product,such as a nutritional supplement.

Generally, the compositions of the invention are for the treatment ofhumans, although they may be used to treat animals including monogastricmammals such as poultry, pigs, cats, dogs, horses or rabbits. Thecompositions of the invention may be useful for enhancing the growth andperformance of animals. If administered to animals, oral gavage may beused.

In some embodiments, the subject to whom the composition is to beadministered is an adult human. In some embodiments, the subject to whomthe composition is to be administered is an infant human.

Compositions

Generally, the composition of the invention comprises bacteria. Inpreferred embodiments of the invention, the composition is formulated infreeze-dried form. For example, the composition of the invention maycomprise granules or gelatin capsules, for example hard gelatincapsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilisedbacteria. Lyophilisation of bacteria is a well-established procedure andrelevant guidance is available in, for example, references [25-27].Theexamples demonstrate that lyophilisate compositions are particularlyeffective.

Alternatively, the composition of the invention may comprise a live,active bacterial culture. The examples demonstrate that cultures of thebacteria of the invention are therapeutically effective.

In some embodiments, the bacterial strain in the composition of theinvention has not been inactivated, for example, has not beenheat-inactivated. In some embodiments, the bacterial strain in thecomposition of the invention has not been killed, for example, has notbeen heat-killed. In some embodiments, the bacterial strain in thecomposition of the invention has not been attenuated, for example, hasnot been heat-attenuated. For example, in some embodiments, thebacterial strain in the composition of the invention has not beenkilled, inactivated and/or attenuated. For example, in some embodiments,the bacterial strain in the composition of the invention is live. Forexample, in some embodiments, the bacterial strain in the composition ofthe invention is viable. For example, in some embodiments, the bacterialstrain in the composition of the invention is capable of partially ortotally colonising the intestine. For example, in some embodiments, thebacterial strain in the composition of the invention is viable andcapable of partially or totally colonising the intestine.

In some embodiments, the composition comprises a mixture of livebacterial strains and bacterial strains that have been killed.

In preferred embodiments, the composition of the invention isencapsulated to enable delivery of the bacterial strain to theintestine. Encapsulation protects the composition from degradation untildelivery at the target location through, for example, rupturing withchemical or physical stimuli such as pressure, enzymatic activity, orphysical disintegration, which may be triggered by changes in pH. Anyappropriate encapsulation method may be used. Exemplary encapsulationtechniques include entrapment within a porous matrix, attachment oradsorption on solid carrier surfaces, self-aggregation by flocculationor with cross-linking agents, and mechanical containment behind amicroporous membrane or a microcapsule. Guidance on encapsulation thatmay be useful for preparing compositions of the invention is availablein, for example, references [28-29].

The composition may be administered orally and may be in the form of atablet, capsule or powder. Encapsulated products are preferred becauseBlautia are anaerobes. Other ingredients (such as vitamin C, forexample), may be included as oxygen scavengers and prebiotic substratesto improve the delivery and/or partial or total colonisation andsurvival in vivo. Alternatively, the probiotic composition of theinvention may be administered orally as a food or nutritional product,such as milk or whey based fermented dairy product, or as apharmaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutically effectiveamount of a bacterial strain of the invention. A therapeuticallyeffective amount of a bacterial strain is sufficient to exert abeneficial effect upon a patient. A therapeutically effective amount ofa bacterial strain may be sufficient to result in delivery to and/orpartial or total colonisation of the patient's intestine.

A suitable daily dose of the bacteria, for example for an adult human,may be from about 1×10³ to about 1×10¹¹ colony forming units (CFU); forexample, from about 1×10⁷ to about 1×10¹⁰ CFU; in another example fromabout 1×10⁶ to about 1×10¹⁰ CFU; in another example from about 1×10⁷ toabout 1×10¹¹ CFU; in another example from about 1×10⁸ to about 1×10¹⁰CFU; in another example from about 1×10⁸ to about 1×10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cellsper day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cellsper day.

In certain embodiments, the composition contains the bacterial strain inan amount of from about 1×10⁶ to about 1×10¹¹ CFU/g, respect to theweight of the composition; for example, from about 1×10⁸ to about 1×10¹⁰CFU/g. The dose may be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, a probiotic, such as the composition of the invention, isoptionally combined with at least one suitable prebiotic compound. Aprebiotic compound is usually a non-digestible carbohydrate such as anoligo- or polysaccharide, or a sugar alcohol, which is not degraded orabsorbed in the upper digestive tract. Known prebiotics includecommercial products such as inulin and transgalacto-oligosaccharides.

In certain embodiments, the probiotic composition of the presentinvention includes a prebiotic compound in an amount of from about 1 toabout 30% by weight, respect to the total weight composition, (e.g. from5 to 20% by weight). Carbohydrates may be selected from the groupconsisting of: fructo-oligosaccharides (or FOS), short-chainfructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins,xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS),beta-glucans, arable gum modified and resistant starches, polydextrose,D-tagatose, acacia fibers, carob, oats, and citrus fibers. In oneaspect, the prebiotics are the short-chain fructo-oligosaccharides (forsimplicity shown herein below as FOSs-c.c); said FOSs-c.c. are notdigestible carbohydrates, generally obtained by the conversion of thebeet sugar and including a saccharose molecule to which three glucosemolecules are bonded.

The compositions of the invention may comprise pharmaceuticallyacceptable excipients or carriers. Examples of such suitable excipientsmay be found in the reference [30]. Acceptable carriers or diluents fortherapeutic use are well known in the pharmaceutical art and aredescribed, for example, in reference [31]. Examples of suitable carriersinclude lactose, starch, glucose, methyl cellulose, magnesium stearate,mannitol, sorbitol and the like. Examples of suitable diluents includeethanol, glycerol and water. The choice of pharmaceutical carrier,excipient or diluent can be selected with regard to the intended routeof administration and standard pharmaceutical practice. Thepharmaceutical compositions may comprise as, or in addition to, thecarrier, excipient or diluent any suitable binder(s), lubricant(s),suspending agent(s), coating agent(s), solubilising agent(s). Examplesof suitable binders include starch, gelatin, natural sugars such asglucose, anhydrous lactose, free-flow lactose, beta-lactose, cornsweeteners, natural and synthetic gums, such as acacia, tragacanth orsodium alginate, carboxymethyl cellulose and polyethylene glycol.Examples of suitable lubricants include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Preservatives, stabilizers, dyes and even flavouring agentsmay be provided in the pharmaceutical composition. Examples ofpreservatives include sodium benzoate, sorbic acid, cysteine and estersof p-hydroxybenzoic acid, for example, in some embodiments thepreservative is selected from sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid. Antioxidants and suspending agents may be alsoused. A further example of a suitable carrier is saccharose. A furtherexample of a preservative is cysteine.

The compositions of the invention may be formulated as a food product.For example, a food product may provide nutritional benefit in additionto the therapeutic effect of the invention, such as in a nutritionalsupplement. Similarly, a food product may be formulated to enhance thetaste of the composition of the invention or to make the compositionmore attractive to consume by being more similar to a common food item,rather than to a pharmaceutical composition. In certain embodiments, thecomposition of the invention is formulated as a milk-based product. Theterm “milk-based product” means any liquid or semi-solid milk- orwhey-based product having a varying fat content. The milk-based productcan be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, wholemilk, milk recombined from powdered milk and whey without anyprocessing, or a processed product, such as yoghurt, curdled milk, curd,sour milk, sour whole milk, butter milk and other sour milk products.Another important group includes milk beverages, such as whey beverages,fermented milks, condensed milks, infant or baby milks; flavoured milks,ice cream; milk-containing food such as sweets.

In some embodiments, the compositions of the invention comprise one ormore bacterial strains of the genus Blautia and do not contain bacteriafrom any other genus, or which comprise only de minimis or biologicallyirrelevant amounts of bacteria from another genus.

In certain embodiments, the compositions of the invention contain asingle bacterial strain or species and do not contain any otherbacterial strains or species. Such compositions may comprise only deminimis or biologically irrelevant amounts of other bacterial strains orspecies. Such compositions may be a culture that is substantially freefrom other species of organism. In some embodiments, such compositionsmay be a lyophilisate that is substantially free from other species oforganism.

In certain embodiments, the compositions of the invention comprise oneor more bacterial strains of the genus Blautia, for example, a Blautiahydrogenotrophica, and do not contain any other bacterial genus, orwhich comprise only de minimis or biologically irrelevant amounts ofbacteria from another genus. In certain embodiments, the compositions ofthe invention comprise a single species of Blautia, for example, aBlautia hydrogenotrophica, and do not contain any other bacterialspecies, or which comprise only de minimis or biologically irrelevantamounts of bacteria from another species. In certain embodiments, thecompositions of the invention comprise a single strain of Blautia, forexample, of Blautia hydrogenotrophica, and do not contain any otherbacterial strains or species, or which comprise only de minimis orbiologically irrelevant amounts of bacteria from another strain orspecies.

In some embodiments, the compositions of the invention comprise morethan one bacterial strain or species. For example, in some embodiments,the compositions of the invention comprise more than one strain fromwithin the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not containbacteria from any other species. In some embodiments, the compositionsof the invention comprise less than 50 strains from within the samespecies (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6,5, 4 or 3 strains), and, optionally, do not contain bacteria from anyother species. In some embodiments, the compositions of the inventioncomprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6,1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15,16-25, or 31-50 strains from within the same species and, optionally, donot contain bacteria from any other species. In some embodiments, thecompositions of the invention comprise more than one species from withinthe same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not containbacteria from any other genus. In some embodiments, the compositions ofthe invention comprise less than 50 species from within the same genus(e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or3 species), and, optionally, do not contain bacteria from any othergenus. In some embodiments, the compositions of the invention comprise1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or31-50 species from within the same genus and, optionally, do not containbacteria from any other genus. The invention comprises any combinationof the foregoing.

In some embodiments, the composition comprises a microbial consortium.For example, in some embodiments, the composition comprises the Blautiabacterial strain as part of a microbial consortium. For example, in someembodiments, the Blautia bacterial strain is present in combination withone or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterialstrains from other genera with which it can live symbiotically in vivoin the intestine. For example, in some embodiments, the compositioncomprises a bacterial strain of Blautia hydrogenotrophica in combinationwith a bacterial strain from a different genus. In some embodiments, themicrobial consortium comprises two or more bacterial strains obtainedfrom a faeces sample of a single organism, e.g. a human. In someembodiments, the microbial consortium is not found together in nature.For example, in some embodiments, the microbial consortium comprisesbacterial strains obtained from faeces samples of at least two differentorganisms. In some embodiments, the two different organisms are from thesame species, e.g. two different humans. In some embodiments, the twodifferent organisms are an infant human and an adult human. In someembodiments, the two different organisms are a human and a non-humanmammal.

In some embodiments, the composition of the invention additionallycomprises a bacterial strain that has the same safety and therapeuticefficacy characteristics as the Blautia hydrogenotrophica straindeposited under accession number DSM 10507/14294, but which is not theBlautia hydrogenotrophica strain deposited under accession number DSM10507/14294, or which is not a Blautia hydrogenotrophica or which is nota Blautia.

In some embodiments in which the composition of the invention comprisesmore than one bacterial strain, species or genus, the individualbacterial strains, species or genera may be for separate, simultaneousor sequential administration. For example, the composition may compriseall of the more than one bacterial strain, species or genera, or thebacterial strains, species or genera may be stored separately and beadministered separately, simultaneously or sequentially. In someembodiments, the more than one bacterial strains, species or genera arestored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention isobtained from human adult faeces. In some embodiments in which thecomposition of the invention comprises more than one bacterial strain,all of the bacterial strains are obtained from human adult faeces or ifother bacterial strains are present they are present only in de minimisamounts. The bacteria may have been cultured subsequent to beingobtained from the human adult faeces and being used in a composition ofthe invention.

In some embodiments, the one or more Blautia bacterial strains is/arethe only therapeutically active agent(s) in a composition of theinvention. In some embodiments, the bacterial strain(s) in thecomposition is/are the only therapeutically active agent(s) in acomposition of the invention.

The compositions for use in accordance with the invention may or may notrequire marketing approval.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is spray dried. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is live. In certain embodiments, the invention providesthe above pharmaceutical composition, wherein the bacterial strain islyophilised or spray dried and wherein it is viable. In certainembodiments, the invention provides the above pharmaceuticalcomposition, wherein the bacterial strain is lyophilised or spray driedand wherein it is capable of partially or totally colonising theintestine. In certain embodiments, the invention provides the abovepharmaceutical composition, wherein the bacterial strain is lyophilisedor spray dried and wherein it is viable and capable of partially ortotally colonising the intestine.

In some cases, the lyophilised or spray dried bacterial strain isreconstituted prior to administration. In some cases, the reconstitutionis by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticallyacceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticalcomposition comprising: a bacterial strain of the invention; and apharmaceutically acceptable excipient, carrier or diluent; wherein thebacterial strain is in an amount sufficient to reduce levels ofEnterobacteriaceae and/or treat a disorder associated with elevatedlevels of Enterobacteriaceae when administered to a subject in needthereof; and wherein the disorder is associated with elevated levels ofEnterobacteriaceae, such as Crohn's disease, ulcerative colitis,functional dyspepsia or, more preferably, IBS, or diarrhoea,gastroenteritis, urinary tract infection or neonatal meningitis.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the amount of the bacterial strain is from about1×10³ to about 1×10¹¹ colony forming units per gram with respect to aweight of the composition.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered at a dose of 1 g, 3g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein the composition is administered by a methodselected from the group consisting of oral, rectal, subcutaneous, nasal,buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a carrier selected from the group consisting oflactose, starch, glucose, methyl cellulose, magnesium stearate, mannitoland sorbitol.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a diluent selected from the group consisting ofethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising an excipient selected from the group consistingof starch, gelatin, glucose, anhydrous lactose, free-flow lactose,beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate,carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate and sodiumchloride.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, further comprising at least one of a preservative, anantioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, comprising a preservative selected from the groupconsisting of sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid.

In certain embodiments, there is provided the pharmaceutical compositionof the invention, wherein the composition does not comprise anyminerals, or more specifically, does not comprise any metals with anatomic number greater than 33, for example, wherein the composition doesnot comprise any minerals from the group consisting of selenium,molybdenum, tungsten, selenium compounds, molybdenum compounds andtungsten compounds.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceuticalcomposition, wherein when the composition is stored in a sealedcontainer at about 4.0 or about 25.0 and the container is placed in anatmosphere having 50% relative humidity, at least 80% of the bacterialstrain as measured in colony forming units, remains after a period of atleast about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years,2.5 years or 3 years.

In some embodiments, the composition of the invention is provided in asealed container comprising a composition as described herein. In someembodiments, the sealed container is a sachet or bottle. In someembodiments, the composition of the invention is provided in a syringecomprising a composition as described herein.

The composition of the present invention may, in some embodiments, beprovided as a pharmaceutical formulation. For example, the compositionmay be provided as a tablet or capsule. In some embodiments, the capsuleis a gelatine capsule (“gel-cap”).

In some embodiments, the compositions of the invention are administeredorally. Oral administration may involve swallowing, so that the compoundenters the gastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth.

Pharmaceutical formulations suitable for oral administration includesolid plugs, solid microparticulates, semi-solid and liquid (includingmultiple phases or dispersed systems) such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids (e.g. aqueoussolutions), emulsions or powders; lozenges (including liquid-filled);chews; gels; fast dispersing dosage forms; films; ovules; sprays; andbuccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an entericformulation, i.e. a gastro-resistant formulation (for example, resistantto gastric pH) that is suitable for delivery of the composition of theinvention to the intestine by oral administration. Enteric formulationsmay be particularly useful when the bacteria or another component of thecomposition is acid-sensitive, e.g. prone to degradation under gastricconditions.

In some embodiments, the enteric formulation comprises an entericcoating. In some embodiments, the formulation is an enteric-coateddosage form. For example, the formulation may be an enteric-coatedtablet or an enteric-coated capsule, or the like. The enteric coatingmay be a conventional enteric coating, for example, a conventionalcoating for a tablet, capsule, or the like for oral delivery. Theformulation may comprise a film coating, for example, a thin film layerof an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric,for example, gastro-resistant without the need for an enteric coating.Thus, in some embodiments, the formulation is an enteric formulationthat does not comprise an enteric coating. In some embodiments, theformulation is a capsule made from a thermogelling material. In someembodiments, the thermogelling material is a cellulosic material, suchas methylcellulose, hydroxymethylcellulose orhydroxypropylmethylcellulose (HPMC). In some embodiments, the capsulecomprises a shell that does not contain any film forming polymer. Insome embodiments, the capsule comprises a shell and the shell compriseshydroxypropylmethylcellulose and does not comprise any film formingpolymer (e.g. see [32]). In some embodiments, the formulation is anintrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, the formulation is a soft capsule. Soft capsulesare capsules which may, owing to additions of softeners, such as, forexample, glycerol, sorbitol, maltitol and polyethylene glycols, presentin the capsule shell, have a certain elasticity and softness. Softcapsules can be produced, for example, on the basis of gelatine orstarch. Gelatine-based soft capsules are commercially available fromvarious suppliers. Depending on the method of administration, such as,for example, orally or rectally, soft capsules can have various shapes,they can be, for example, round, oval, oblong or torpedo-shaped. Softcapsules can be produced by conventional processes, such as, forexample, by the Scherer process, the Accogel process or the droplet orblowing process.

Culturing Methods

The bacterial strains for use in the present invention can be culturedusing standard microbiology techniques as detailed in, for example,references [33-35].

The solid or liquid medium used for culture may for example be YCFA agaror YCFA medium. YCFA medium may include (per 100 ml, approximatevalues): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g),cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g),(NH₄)₂SO₄ (0.09 g), MgSO₄.7H₂O (0.009 g), CaCl₂) (0.009 g), resazurin(0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoicacid (3 μg), folic acid (5 μg), and pyridoxamine (15

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references[36-43], etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

References to a percentage sequence identity between two nucleotidesequences means that, when aligned, that percentage of nucleotides arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref[44]. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. [45].

Unless specifically stated, a process or method comprising numeroussteps may comprise additional steps at the beginning or end of themethod, or may comprise additional intervening steps. Also, steps may becombined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will beappreciated that the features specified in each embodiment may becombined with other specified features, to provide further embodiments.In particular, embodiments highlighted herein as being suitable, typicalor preferred may be combined with each other (except when they aremutually exclusive).

MODES FOR CARRYING OUT THE INVENTION Example 1—Administration ofBacterial Inocula to Rats Harbouring IBS Gut Microbiota Summary

Rats were inoculated with the faecal microbiota from a human IBSsubject. The rats were then administered with compositions comprisingbacterial strains according to the invention and colonisation by Blautiahydrogenotrophica was analysed.

Strain

Blautia hydrogenotrophica (BH) strain DSM 10507/14294.

Compositions and Administration

BH lyophilisate—administered by oral gavage

Control solution administered by oral gavage

Rats

Inoculated with human intestinal microbiota from an IBS subject.

Study Design

Day −14—rats inoculated with human intestinal microbiota from an IBSsubject

Days 0 to 28—daily dose of BH lyophilisate, or control solution

Days 0, 14 and 28—qPCR of BH population in faecal samples

Results

FIG. 1 presents the results of a qPCR analysis of the BH population infaecal samples from rats administered control solution (IBS) or BHlyophilisate (IBS+BH). An increase in the BH population was seen at days14 and 28 in rats receiving the BH lyophilisate, which confirmssuccessful colonisation.

Conclusions

Administration of compositions comprising Blautia hydrogenotrophica ledto successful colonisation.

Example 2—Administration of Bacterial Lyophilisate to Healthy Rats andEffect on Enterobacteriaceae

The effects of administration of a lyophilisate of Blautiahydrogenotrophica (BH) strain DSM 10507/14294 on healthy HIM rats werestudied and the results are reported in FIGS. 2-5. Further detailsregarding the experiments are provided above in the descriptions of thefigures. FIG. 2 shows that an appropriate dose for BH in rats is 10⁹cells per day or greater. FIG. 3 shows that in these experiments BH didnot permanently colonise the rat digestive tract. FIG. 4 shows that BHis primarily found in the caecum. Strikingly, FIG. 5 shows thatadministration of BH induces a statistically-significant reduction inEnterobacteriaceae in rat faeces as detected by qPCR.

Example 3—Administration of Bacterial Lyophilisate in a Rat Model of IBS

The effects of administration of a lyophilisate of Blautiahydrogenotrophica (BH) strain DSM 10507/14294 on a rat model of IBS werefurther investigated. Germ-free rats were inoculated with faecal samplesfrom C-IBS (with constipation) or U-IBS (unsubtyped) patients. Theresults are reported in FIG. 6 and further details regarding theexperiments are provided above in the descriptions of the figures. FIG.6 confirms that administration of BH lyophilisate causes astatistically-significant reduction in Enterobacteriaceae, which ismainly E. coli. As expected, an increase in BH is also observed.

Example 4—Comparison of Patient Microbiota Blautia hydrogenotrophica

A Phase I clinical trial was conducted in which Blautiahydrogenotrophica (“Blautix”, strain deposited under accession numberDSM 10507/14294) was administered to human patients having irritablebowel syndrome (IBS). Patients were administered Blautix during a dosingperiod (day 1-16) with the washout period being day 19-23. Blautix wasfound to be both safe and well tolerated. Patient microbiota Blautiahydrogenotrophica (Blautix) were measured at day 1, day 16 and at theend of the study (EOS) with results as follows:

TABLE 1 Blautix Day 1 Blautix Day 16 Blautix EOS Patient Treatment MeanSD Mean SD Mean SD Placebo Healthy 4.30E+07 8.40E+07 4.79E+07 7.63E+073.59E+07 6.23E+07 Blautix Healthy 2.92E+07 2.25E+07 6.35E+07 1.02E+082.58E+07 3.34E+07 Placebo IBS 3.58E+07 3.45E+07 2.44E+07 3.13E+072.09E+07 2.47E+07 Blautix IBS 3.17E+07 6.28E+07 8.53E+07 1.57E+083.71E+07 5.56E+07

Analysis shows trends in Blautia hydrogenotropica levels in patientstool samples (see FIG. 7A). Participants receiving Blautix haveincreased levels of the bacteria in their stool samples following thedosing period (day 16). This demonstrates the successful delivery ofBlautix to the gut. The levels return to normal following the washoutperiod (End-of study; EOS). Thus Blautix does not permanently colonisethe gut once dosing ceases. Both observations are consistent with thepreclinical model.

Example 5—Comparison of Patient Microbiota Enterobacteria

Patient microbiota Enterobacteria were also measured during the Phase Iclinical trial at day 1, day 16 and at the end of the study with resultsas follows:

Enterobacteria Day 1 Enterobacteria Day 16 Enterobacteria EOS PatientTreatment Mean SD Mean SD Mean SD Placebo Healthy 5.89E+08 1.00E+093.32E+08 6.26E+08 2.85E+08 4.28E+08 Blautix Healthy 9.96E+08 1.06E+094.87E+08 6.64E+08 2.70E+08 3.76E+08 Placebo IBS 7.85E+08 1.28E+093.01E+08 4.92E+08 2.45E+09 2.45E+09 Blautix IBS 8.38E+09 1.96E+101.12E+09 1.45E+09 6.17E+08 9.91E+08

Analysis shows trends in Enterobacteria levels in patients stool samples(see FIG. 7B). IBS patients treated with Blautix had a reduction inEnterobacteria. This is consistent with observations from thepreclinical model.

Example 6—Hydrogen Breath Test Results

Breath hydrogen levels are a biomarker of Blautix activity—MoA involvesmetabolism of endogenous H₂ to produce acetate. Human subjects wereadministered lactulose and hydrogen (H₂) levels (Cmax) were sampled atfour time points: day 1, day 2, day 15 and day 16. Hydrogen uncorrectedresults were converted to hydrogen corrected results.

Some patients were excluded from the analysis. There were three reasonswhy a subject was not included in the hydrogen breath test analysis: 1)They produced a CMAX hydrogen breath test result of <20 on one of thefour sampling days and were therefore deemed to have not responded tothe test; 2) They were methane producers (taken as producing moremethane than hydrogen in the breath test), this affects the hydrogenresponse; and/or 3) there was an aberrant value obtained in the hydrogenbreath test (232 ppm). Subjects 3.12 (Blautix), 3.24 (Blautix), 4.07(Blautix) were excluded as non-responders. Subjects 3.03 (Blautix) and3.08 (Placebo) were excluded as methane producers (4.07, mentioned asexcluded above, was also a methane producer). Subject 4.09 (Placebo) wasexcluded due to an aberrant value.

The results of the corrected hydrogen analysis from the end of thedosing period (day 15/16) were compared to those from the baseline (day1/2). 10 out of 12 patients (83%) receiving Blautix had a reduction inhydrogen levels over this period (FIGS. 8A and 8C). In contrast, 3 outof 6 (50%) patients receiving placebo had reduced hydrogen levels (FIGS.8B and 8C).

FIGS. 9A-9C show the uncorrected and corrected hydrogen results for theBlautix (Verum) treatment group together with a statistical analysis ofthe results. The mean values for both uncorrected and corrected H₂ werefound to differ between day 1 and day 15. After 13.5 days of treatment,a statistically significant (p<0.05) decrease of H₂ in Cmax breath testwas detected after lactulose stimulation. In contrast, for the placebogroup, the mean was found to be equivalent for day 1 and day 15 (p>0.05)(FIGS. 10A-10C). Thus, the mean for the treatment group (also referredto as the VERUM group), decreases between day 1 and day 15 whereas themean for the placebo group is equivalent between day 1 and day 15 forboth the uncorrected hydrogen results and the corrected hydrogen results(FIGS. 11A-11B).

Example 7—Stability Testing

A composition described herein containing at least one bacterial straindescribed herein is stored in a sealed container at 25° C. or 4° C. andthe container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%,75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, atleast 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain asmeasured in colony forming units determined by standard protocols.

Example 8—the Ability of B. hydrogenotrophica to Alter the Microbiota inHuman Microbiota Associated Rat (HMA Rat) Model Summary

Groups of 16 germ-free rats (comprising 8 rats in the control group and8 rats in the treatment group) were inoculated with the faecalmicrobiota from a human IBS subject (IBS-HMA rats). Three successiveexperiments were carried out using faecal samples from 3 different IBSpatients. Two other groups of rats (n=10) were inoculated with faecalsamples of healthy subject (n=2 subjects; 2 groups of healthy-HMA rats)as a control. Thus, there were 24 IBS-microbiota associated rats(control), 24 IBS microbiota associated rats treated with Blautix and 20healthy-microbiota associated rats. Half of the IBS-HMA rats were thenadministered for 28 days with a composition comprising the bacterialstrain of B. hydrogenotrophica according to the invention, while theother half animals received a control solution. Day 28 afteradministration, the levels of microorganisms from faecal microbiota,previously found to be affected in IBS patients was analysed.

Strain

Blautia hydrogenotrophica (BH) strain DSM 10507^(T)/14294.

Composition and Administration

BH lyophilisate was suspended in sterile mineral solution to aconcentration of 10¹⁰ bacteria per ml. Two ml of this suspension wasadministered daily per IBS-HMA rat, by oral gavage, for a 28 daysperiod.

The control solution was the sterile mineral solution that wasadministered daily (2 ml per rat) by oral gavage to the control group ofIBS-HMA rats.

Rats

Germ-Free male Fisher rats (aged 10 weeks) were inoculated with humanfaecal microbiota from an IBS subject (IBS-HMA rats). Sixteen rats wereinoculated with the same human faecal inoculum. Three successiveexperiments were performed with faecal samples from three different IBSsubjects. Two other groups of ten rats were inoculated with faecalsample from 2 healthy subjects (normo-sensitivity control groups).

Study Design

Day −14—Inoculation of Germ-free rats with human faecal microbiota.

Days 0 to 28—Daily dose of BH lyophilisate (assay group), or controlsolution (control group) by oral gavage

Days 0, 14 and 28—Collection of faecal samples for microbial analysis:qPCR for evaluating BH population and other commensal groups ofmiccroorganisms and enumeration of functional groups of microorganismsusing selective media and strictly anaerobic method.

Results

FIG. 12 presents the results of qPCR analysis of the B.hydrogenotrophica population in faecal samples from IBS-HMA ratsreceiving control solution or BH lyophilisate. A significant increase inthe BH population was observed at the end of the administration period(D 28) in rats receiving the BH lyophilisate, which confirms successfuldelivery of BH in the colon.

FIG. 13 reports on effect of administration of B. hydrogenotrophica oncertain microorganisms in faecal microbiota, previously found to beaffected in IBS patients. The level of Enterobacteriaceae speciesdecreased after B. hydrogenotrophica administration.

Conclusions

Administration of Blautia hydrogenotrophica resulted in a reduction inEnterobacteriaceae.

Example 9—Changes in Patient Symptoms During Phase I Clinical Trial

A Phase I clinical trial was conducted in which Blautiahydrogenotrophica (“Blautix”, strain deposited under accession numberDSM 10507 and also under accession number DSM 14294) was administered tohuman patients having irritable bowel syndrome (IBS). Patients wereadministered Blautix during a dosing period (days 1-16) with the washoutperiod being day 19-23. Blautix was found to be both safe and welltolerated. Four symptoms were monitored, of which one was diarrhoea. Thestudy recorded whether patients experienced an improvement in, no changein or worsening of each of these symptoms. Results from patientsadministered Blautix were compared with those obtained using patientsadministered a placebo. Symptoms were monitored at three time points:day 1, day 15/16 and at the end of the study. The results are shown inFIGS. 14 and 15.

When the patients' reported symptoms at day 16 were compared to thebaseline from day 1, 82% of 17 IBS patients receiving Blautix reportedan improvement in symptoms (FIG. 14). Improvement of symptoms, of whichone was diarrhoea, supports the use of Blautix for treating orpreventing diarrhoea.

50% of patients receiving placebo reported an improvement in symptoms(FIG. 14). High placebo response rates are an established phenomenon inIBS clinical studies. Xifaxan was recently approved to treat IBS basedon much smaller improvements over placebo (see:http://www.accessdata.fda.gov/spl/data/5ab6fceb-4d22-4480-81fc-8bc28c16770d/5ab6fceb-4d22-4480-81fc-8bc28c16770d.xml).

A worsening of symptoms at the study completion (day 19-23) compared tosymptoms present upon dosing completion (day 16) is expected based onthe teaching presented here. This worsening of symptoms was seen in thePhase I clinical trial: 41% of IBS patients reported worsening ofsymptoms following cessation of Blautix dosing (FIG. 15). The worseningof symptoms, one of which is diarrhoea, following cessation of Blautixdosing therefore also supports the use of Blautix in treating orpreventing diarrhoea.

Sequences(Blautia stercoris strain GAM6-1 16S ribosomal RNA gene, partial sequence -HM626177) SEQ ID NO: 1   1 tgcaagtcga gcgaagcgct tacgacagaa ccttcggggg aagatgtaag ggactgagcg  61 gcggacgggt gagtaacgcg tgggtaacct gcctcataca gggggataac agttggaaac 121 ggctgctaat accgcataag cgcacggtat cgcatgatac agtgtgaaaa actccggtgg 181 tatgagatgg acccgcgtct gattagctag ttggaggggt aacggcccac caaggcgacg 241 atcagtagcc ggcctgagag ggtgaacggc cacattggga ctgagacacg gcccagactc 301 ctacgggagg cagcagtggg gaatattgca caatggggga aaccctgatg cagcgacgcc 361 gcgtgaagga agaagtatct cggtatgtaa acttctatca gcagggaaga aaatgacggt 421 acctgactaa gaagccccgg ctaactacgt gccagcagcc gcggtaatac gtagggggca 481 agcgttatcc ggatttactg ggtgtaaagg gagcgtagac ggaagagcaa gtctgatgtg 541 aaaggctggg gcttaacccc aggactgcat tggaaactgt ttttcttgag tgccggagag 601 gtaagcggaa ttcctagtgt agcggtgaaa tgcgtagata ttaggaggaa caccagtggc 661 gaaggcggct tactggacgg taactgacgt tgaggctcga aagcgtgggg agcaaacagg 721 attagatacc ctggtagtcc acgccgtaaa cgatgaatac taggtgttgg ggagcaaagc 781 tcttcggtgc cgcagcaaac gcaataagta ttccacctgg ggagtacgtt cgcaagaatg 841 aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag 901 caacgcgaag aaccttacca agtcttgaca tcgatctgac cggttcgtaa tggaaccttt 961 ccttcgggac agagaagaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt1021 gggttaagtc ccgcaacgag cgcaacccct atcctcagta gccagcaggt gaagctgggc1081 actctgtgga gactgccagg gataacctgg aggaaggcgg ggacgacgtc aaatcatcat1141 gccccttatg atttgggcta cacacgtgct acaatggcgt aaacaaaggg aagcgagccc1201 gcgaggggga gcaaatccca aaaataacgt cccagttcgg actgcagtct gcaactcgac1261 tgcacgaagc tggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg1321 ggtcttgtac acaccgcccg tcacaccatg ggagtcagta acgcccgaag tc(Blautia wexlerae strain WAL 14507 16S ribosomal RNA gene, partialsequence - EF036467) SEQ ID NO: 2   1 caagtcgaac gggaattant ttattgaaac ttcggtcgat ttaatttaat tctagtggcg  61 gacgggtgag taacgcgtgg gtaacctgcc ttatacaggg ggataacagt cagaaatggc 121 tgctaatacc gcataagcgc acagagctgc atggctcagt gtgaaaaact ccggtggtat 181 aagatggacc cgcgttggat tagcttgttg gtggggtaac ggcccaccaa ggcgacgatc 241 catagccggc ctgagagggt gaacggccac attgggactg agacacggcc cagactccta 301 cgggaggcag cagtggggaa tattgcacaa tgggggaaac cctgatgcag cgacgccgcg 361 tgaaggaaga agtatctcgg tatgtaaact tctatcagca gggaagatag tgacggtacc 421 tgactaagaa gccccggcta actacgtgcc agcagccgcg gtaatacgta gggggcaagc 481 gttatccgga tttactgggt gtaaagggag cgtagacggt gtggcaagtc tgatgtgaaa 541 ggcatgggct caacctgtgg actgcattgg aaactgtcat acttgagtgc cggaggggta 601 agcggaattc ctagtgtagc ggtgaaatgc gtagatatta ggaggaacac cagtggcgaa 661 ggcggcttac tggacggtaa ctgacgttga ggctcgaaag cgtggggagc aaacaggatt 721 agataccctg gtagtccacg ccgtaaacga tgaataacta ggtgtcgggt ggcaaagcca 781 ttcggtgccg tcgcaaacgc agtaagtatt ccacctgggg agtacgttcg caagaatgaa 841 actcaaagga attgacgggg acccgcacaa gcggtggagc atgtggttta attcgaagca 901 acgcgaagaa ccttaccaag tcttgacatc cgcctgaccg atccttaacc ggatctttcc 961 ttcgggacag gcgagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg1021 gttaagtccc gcaacgagcg caacccctat cctcagtagc cagcatttaa ggtgggcact1081 ctggggagac tgccagggat aacctggagg aaggcgggga tgacgtcaaa tcatcatgcc1141 ccttatgatt tgggctacac acgtgctaca atggcgtaaa caaagggaag cgagattgtg1201 agatggagca aatcccaaaa ataacgtccc agttcggact gtagtctgca acccgactac1261 acgaagctgg aatcgctagt aatcgcggat cagaatgccg cggtgaatac gttcccgggt1321 cttgtacaca ccgcccgtca caccatggga gtcagtaacg cccgaagtca gtgacctaac1381 tgcaaagaag gagctgccga aggcgggacc gatgactggg gtgaagtcgt aacaaggt(consensus 16S rRNA sequence for Blautia stercoris strain 830)SEQ ID NO: 3TTTKGTCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCGCTTACGACAGAACCTTCGGGGGAAGATGTAAGGGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTGGAAACGGCTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTTGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCGATCTGACCGGTTCGTAATGGAACCTTTCCTTCGGGACAGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCGTCAGTAGCCAGCAGGTAAAGCTGGGCACTCTGAGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGCCCGCGAGGGGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTAGGGAGGGAGCTGCCGAAGGCGGGATTGATAACTGGGGTGAAGTCTAGGGGGT(consensus 16S rRNA sequence for Blautia wexlerae strain MRX008)SEQ ID NO: 4TTCATTGAGACTTCGGTGGATTTAGATTCTATTTCTAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTATACAGGGGGATAACAGTCAGAAATGGCTGCTAATACCGCATAAGCGCACAGAGCTGCATGGCTCAGTGTGAAAAACTCCGGTGGTATAAGATGGACCCGCGTTGGATTAGCTTGTTGGTGGGGTAACGGCCCACCAAGGCGACGATCCATAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGATAGTGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTGTGGCAAGTCTGATGTGAAAGGCATGGGCTCAACCTGTGGACTGCATTGGAAACTGTCATACTTGAGTGCCGGAGGGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCNGGGGAGCATGGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCGCCTGACCGATCCTTAACCGGATCTTTCCTTCGGGACAGGCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTCAGTAGCCAGCATTTAAGGTGGGCACTCTGGGGAGACTGCCAGGGATAACCTGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGATCGTGAGATGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCTAACTGCAAAGAAGGAGCTGCCGAA(Blautia hydrogenotrophica strain S5a36 16S ribosomal RNA gene, partialsequence - X95624.1) SEQ ID NO: 5   1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gaagcgatag agaacggaga  61 tttcggttga agttttctat tgactgagtg gcggacgggt gagtaacgcg tgggtaacct 121 gccctataca gggggataac agttagaaat gactgctaat accgcataag cgcacagctt 181 cgcatgaagc ggtgtgaaaa actgaggtgg tataggatgg acccgcgttg gattagctag 241 ttggtgaggt aacggcccac caaggcgacg atccatagcc ggcctgagag ggtgaacggc 301 cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg gaatattgca 361 caatggggga aaccctgatg cagcgacgcc gcgtgaagga agaagtatct cggtatgtaa 421 acttctatca gcagggaaga aagtgacggt acctgactaa gaagccccgg ctaattacgt 481 gccagcagcc gcggtaatac gtaaggggca agcgttatcc ggatttactg ggtgtaaagg 541 gagcgtagac ggtttggcaa gtctgatgtg aaaggcatgg gctcaacctg tggactgcat 601 tggaaactgt cagacttgag tgccggagag gcaagcggaa ttcctagtgt agcggtgaaa 661 tgcgtagata ttaggaggaa caccagtggc gaaggcggcc tgctggacgg taactgacgt 721 tgaggctcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa 781 cgatgaatac taggtgtcgg gtggcaaagc cattcggtgc cgcagcaaac gcaataagta 841 ttcccacctg gggagtacgt tcgcaagaat gaaactcaaa ggaattgacg gggacccgca 901 caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aaatcttgac 961 atccctctga ccgggaagta atgttccctt ttcttcggaa cagaggagac aggtggtgca1021 tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct1081 tattcttagt agccagcagg tagagctggg cactctaggg agactgccag ggataacctg1141 gaggaaggtg gggatgacgt caaatcatca tgccccttat gatttgggct acacacgtgc1201 tacaatggcg taaacaaagg gaagcgaagg ggtgacctgg agcaaatctc aaaaataacg1261 tctcagttcg gattgtagtc tgcaactcga ctacatgaag ctggaatcgc tagtaatcgc1321 gaatcagaat gtcgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccat1381 gggagtcagt aacgcccgaa gtcagtgacc caaccnaaag gagggagctg ccgaaggtgg1441 gactgataac tggggtga

REFERENCES

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1.-26. (canceled)
 27. A method of treating a systemic condition in asubject in need thereof, comprising administering to said subject apharmaceutical composition that comprises at least 1×10³ colony formingunits (CFU) per gram of a bacteria strain of the genus Blautia withrespect to a total weight of said pharmaceutical composition, whereinsaid bacteria strain comprises a 16S rRNA gene having at least 80%sequence identity to the polynucleotide sequence of SEQ ID NO:5, asdetermined by a Smith-Waterman homology search algorithm using an affinegap search with a gap open penalty of 12, a gap extension penalty of 2,and a Blocks Substitution Matrix (BLOSUM) of 62, wherein saidadministering is effective to treat said systemic condition in saidsubject.
 28. The method of claim 27, wherein said systemic condition isa urinary tract infection.
 29. The method of claim 27, wherein saidsystemic condition is neonatal meningitis.
 30. The method of claim 27,wherein said pharmaceutical composition comprises from about 1×10³ toabout 1×10¹¹ CFU/g of said bacteria strain with respect to a totalweight of said pharmaceutical composition.
 31. The method of claim 27,wherein said pharmaceutical composition comprises from about 1×10⁹ toabout 1×10¹² CFU of said bacteria strain.
 32. The method of claim 27,further comprising administering an antibiotic treatment.
 33. The methodof claim 32, wherein said administering of said antibiotic treatment isconcurrent with said administering of said pharmaceutical composition.34. The method of claim 32, wherein said antibiotic is fluoroquinolone,azithromycin, ciprofloxacin or rifaximin.
 35. The method of claim 27,further comprising performing dehydration therapy to said subject. 36.The method of claim 27, wherein said subject is a human.
 37. The methodof claim 27, wherein said bacteria strain comprises a 16S rRNA genehaving at least 95% sequence identity to the polynucleotide sequence ofSEQ ID NO:5, as determined by said Smith-Waterman homology searchalgorithm using said affine gap search with said gap open penalty of 12,said gap extension penalty of 2, and said Blocks Substitution Matrix(BLOSUM) of
 62. 38. The method of claim 27, wherein said administeringof said pharmaceutical composition is oral.
 39. The method of claim 27,wherein said administering of said pharmaceutical composition is rectal.40. The method of claim 39, wherein said pharmaceutical composition isformulated as a suppository.
 41. The method of claim 40, wherein saidsuppository comprises cocoa butter, suppocire, witepsol,glycero-gelatin, polyethylene glycol, or soap glycerin.
 42. The methodof claim 27, wherein said bacteria strain is dried.
 43. The method ofclaim 27, wherein said pharmaceutical composition further comprises alyoprotectant.
 44. The method of claim 27, wherein said pharmaceuticalcomposition further comprises an enteric coating.
 45. The method ofclaim 27, wherein said pharmaceutical composition is a capsuleformulation.
 46. The method of claim 27, wherein said bacteria strain isof the species Blautia hydrogenotrophica.